人b干扰素与人血清白蛋白融合蛋白的表达及纯化

Expression and purification of IFNβ-HSA fusion protein in Pichia pastoris

摘要:

为开展人b干扰素-人血清白蛋白融合蛋白(IFNb-HAS)的临床前研究, 本研究采用免疫学方法对毕赤酵母转化子进行筛选, 得到高产菌株8株。在5 L发酵罐上对高产菌株进行诱导表达, 产量达500 mg/L。发酵液经超滤浓缩、染料亲和层析、镍离子螯合亲和层析和DEAE离子交换层析纯化后, 融合蛋白纯度达到96%。Western blotting杂交表明融合蛋白具有人血清白蛋白和人b干扰素的抗原性, 细胞病变抑制法测定IFNb-HSA比活性为1.96×107 IU/mg。建立了融合蛋白的生产工艺, 为IFNb-HSA的临床前研究奠定了基础。

Abstract:

In order to obtain enough fusion protein for developing preclinical studies of IFNb-HAS, we screened Pichia pastoris transformants expressing high-level protein by immunology method. The yield of IFNβ-HSA was about 500 mg/L by fed-batch fermentation. The purity of IFNβ-HSA reached 96% through the steps of ultrafiltration, Blue Sepharose FF, Ni2+-IMAC and DEAE Sepharose FF. Analysis of Western blotting showed that IFNβ-HSA had the antigenicity of IFNβ and HSA. The specific activity was about 1.96×107 IU/mg by standard survival activity test on WISH cells challenged with VSV virus. This study provided a method to produce IFNβ-HSA.